Solvent-Free Microfluidic Fabrication of Antimicrobial Lipid Nanoparticles

Antimicrobial lipid nanoparticles composed of monoglycerides offer a promising strategy to inhibit membrane-enveloped viral and bacterial pathogens. However, previous efforts mainly focused on fabricating nanoparticles from long-chain monoglycerides, which lack intrinsic antimicrobial activity but contribute to nanoparticle stability and structural integrity. In contrast, shorter-chain monoglycerides often exhibit potent antimicrobial effects but do not self-assemble into colloidally stable nanoparticles and lose efficacy upon dilution. To overcome these limitations and incorporate antimicrobial monoglycerides into a stable nanoparticle configuration, we report a solvent-free microfluidic fabrication strategy that combines the functional characteristics of different monoglycerides to prepare interfacially active, monoglyceride-based nanoparticles with mixed compositions that display potent antibacterial activity. Unlike conventional microfluidic mixing methods that rely on volatile organic solvents, our approach utilizes pharmaceutical-grade materials and does not require organic solvent removal, hence eliminating the need for a dialysis step postfabrication. Dynamic light scattering (DLS) and zeta potential measurements verified that the fabricated nanoparticles had ∼250-350 nm diameters and exhibited high colloidal stability whereas the antibacterial activity of the nanoparticles against <i>Staphylococcus aureus</i> bacteria depended strongly on the nanoparticle composition. Nanoparticles composed of glycerol monooleate alone were inactive, while the inclusion of glycerol monolaurate slightly enhanced antibacterial activity. Surprisingly, the further addition of glycerol monobehenate or glycerol dibehenate─previously considered inactive structural components that are used to improve nanoparticle cohesion─boosted antibacterial potency by up to 270-fold. Biophysical experiments showed that nanoparticle compositions with greater antibacterial activity induced more pronounced membrane disruption, as observed in quartz crystal microbalance-dissipation and electrochemical impedance spectroscopy measurements. These findings demonstrate that combining different monoglycerides can significantly enhance the antibacterial activity of lipid-based nanoparticles and underscore the potential of membrane biophysics approaches to guide performance optimization, highlighting the capability to tune membrane-disruptive properties in physiologically relevant pH conditions.